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Epithelial-to-mesenchymal transitions (EMTs) are phenotypic plasticity processes that confer migratory and invasive properties to epithelial cells during development, wound-healing, fibrosis and cancer 1 – 4 . EMTs are driven by SNAIL, ZEB and TWIST transcription factors 5 , 6 together with microRNAs that balance this regulatory network 7 , 8 . Transforming growth factor β (TGF-β) is a potent inducer of developmental and fibrogenic EMTs 4 , 9 , 10 . Aberrant TGF-β signalling and EMT are implicated in the pathogenesis of renal fibrosis, alcoholic liver disease, non-alcoholic steatohepatitis, pulmonary fibrosis and cancer 4 , 11 . TGF-β depends on RAS and mitogen-activated protein kinase (MAPK) pathway inputs for the induction of EMTs 12 – 19 . Here we show how these signals coordinately trigger EMTs and integrate them with broader pathophysiological processes. We identify RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector 20 , 21 , as a key partner of TGF-β-activated SMAD transcription factors in EMT. MAPK-activated RREB1 recruits TGF-β-activated SMAD factors to SNAIL . Context-dependent chromatin accessibility dictates the ability of RREB1 and SMAD to activate additional genes that determine the nature of the resulting EMT. In carcinoma cells, TGF-β–SMAD and RREB1 directly drive expression of SNAIL and fibrogenic factors stimulating myofibroblasts, promoting intratumoral fibrosis and supporting tumour growth. In mouse epiblast progenitors, Nodal–SMAD and RREB1 combine to induce expression of SNAIL and mesendoderm-differentiation genes that drive gastrulation. Thus, RREB1 provides a molecular link between RAS and TGF-β pathways for coordinated induction of developmental and fibrogenic EMTs. These insights increase our understanding of the regulation of epithelial plasticity and its pathophysiological consequences in development, fibrosis and cancer. RAS and TGF-β pathways regulate distinct modes of epithelial-to-mesenchymal transition via RAS-responsive element binding protein 1.

Mitochondrial DNA stress potentiates type I interferon responses via activation of the cGAS–STING–IRF3 pathway. Mitochondria trigger innate immunity Accumulating evidence suggests that mitochondria, the organelles primarily responsible for cellular respiration and energy production, are also critical centres for antibacterial and antiviral innate immune responses. This study describes a link between mitochondrial stress and antiviral innate immunity. Specifically, mitochondrial DNA stress in herpes virus infected mice is shown to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance through activation of the DNA sensor cGAS and the STING–IRF3 stress pathway. Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids 1 . The abundant mtDNA-binding protein TFAM (transcription factor A, mitochondrial) regulates nucleoid architecture, abundance and segregation 2 . Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses 3 . However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined 4 . Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signalling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

A basic leucine zipper transcription factor, NF-E2-related factor 2 (Nrf2), plays a critical role in the cellular defense mechanism by mediating a coordinate up-regulation of antioxidant responsive element-driven detoxification and antioxidant genes. Here, we report that targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia due to increased sequestration of damaged erythrocytes. Splenomegaly and spleen toxicity in Nrf2-/-mice raised a possibility of hemolytic anemia and splenic extramedullary hematopoiesis in Nrf2-/-mice. In support of this, hematology analysis revealed that Nrf2-/-mice suffer from anemia with abnormal red cell morphologies (i.e., Howell-Jolly bodies, acantocytes, and schistocytes). In addition, Nrf2-/-erythrocytes were more sensitive to H2O2-induced hemolysis, and erythrocytebound IgG levels were markedly increased in Nrf2-/-mice compared with Nrf2+/+mice. Because IgG bound to erythrocytes in the presence of oxidative damage in erythrocytes (regardless of Nrf2 genotype), these data support that Nrf2-/-erythrocytes have higher levels of damage compared with Nrf2+/+cells. Finally, Nrf2-/-mice showed increased levels of erythrocyte-bound IgG compared with Nrf2+/+mice after H2O2injection in vivo, suggesting that the decreased glutathione and increased H2O2render the Nrf2-/-mice more susceptible to toxicity. Taken together, these observations indicate that a chronic increase in oxidative stress due to decreased antioxidant capacity sensitizes erythrocytes and causes hemolytic anemia in Nrf2-/-mice, suggesting a pivotal role of Nrf2- antioxidant responsive element pathway in the cellular antioxidant defense system.

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies 1 , 2 . CAR T cells have been less effective against solid tumours 3 – 5 , in part because they enter a hyporesponsive (‘exhausted’ or ‘dysfunctional’) state 6 – 9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19 + tumour-bearing mice. CD8 + CAR + tumour-infiltrating lymphocytes and CD8 + endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells) 10 – 12 . CD8 + T cells from humans with cancer or chronic viral infections 13 – 15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors ( Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8 + effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy. Transfer of NR4A-deficient T cells expressing chimeric antigen receptors is shown to reduce tumour burden and increase survival by shifting T cell transcriptional programs away from exhaustion and towards increased effector function.

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro . In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylation. Genes silenced by hypoxia Tumours are epigenetically distinct from their tissue of origin, frequently showing increased DNA methylation of tumour suppressor gene promoters, but how these changes arise is poorly understood. Here, Diether Lambrechts and colleagues report that tumour hypoxia, pervasive in many solid tumours, reduces the activity of the oxygen-dependent ten-eleven translocation (TET) enzymes, which catalyse DNA demethylation through 5-methylcytosine oxidation. They show that oxygen is an important co-factor for TET activity, and hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro and at tumour suppressor genes in hypoxic tumours. The authors propose that tumour hypoxia directly reduces TET activity, leading to changes in DNA methylation and silencing gene expression. Countering hypermethylation by inhibiting DNA methylation or by normalizing tumour blood supply may therefore be of therapeutic benefit.

Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression 1 . However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A–SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ 2 . Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A–SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A–SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A–SWI/SNF, thereby preventing the formation of the ARID1A–SWI/SNF–YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A–SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis. The ARID1A-containing SWI/SNF complex operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ; this interaction is regulated by cellular mechanotransduction.

The CXXC domains of TET2 (encoded by the distinct gene IDAX ) and TET3 are found to have previously unknown roles in the regulation of TET proteins through the activation of caspases and subsequent reduction in TET catalytic activity; this regulation is dependent on DNA binding through the CXXC domain. IDAX regulates TET2 protein expression TET family proteins modify the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC, sometimes called the 'fifth base' of DNA) and other intermediates. TET1 and TET3 contain a CXXC domain but the ancestral CXXC domain of TET2 is encoded by a distinct gene, IDAX (or CXXC4 ). This paper demonstrates that IDAX binds unmethylated CpG-rich DNA via its CXXC domain and recruits TET2. The separate and linked CXXC domains of TET2 and TET3 are shown to act as regulators of caspase activation and TET enzymatic activity. The authors suggest that future studies should focus on the genomic targets of TET2, IDAX and the IDAX-related protein CXXC5 in normal development and in cancer. TET (ten-eleven-translocation) proteins are Fe( ii )- and α-ketoglutarate-dependent dioxygenases 1 , 2 , 3 that modify the methylation status of DNA by successively oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine 1 , 3 , 4 , 5 , potential intermediates in the active erasure of DNA-methylation marks 5 , 6 . Here we show that IDAX (also known as CXXC4), a reported inhibitor of Wnt signalling 7 that has been implicated in malignant renal cell carcinoma 8 and colonic villous adenoma 9 , regulates TET2 protein expression. IDAX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The IDAX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. Unexpectedly, IDAX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the IDAX CXXC domain, suggesting that IDAX recruits TET2 to DNA before degradation. IDAX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against IDAX increases TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 is also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3, respectively, as previously unknown regulators of caspase activation and TET enzymatic activity.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling. This study shows that 53BP1 recruitment to sites of DNA damage involves dual recognition of H4K20me2 and H2AK15 histone ubiquitination; the ubiquitin mark and the surrounding epitope on H2A are read by a region of 53BP1 designated the ubiquitination-dependent recruitment motif. Recruiting 53BP1 protein to DNA damage sites The key DNA damage response protein 53BP1 acts by binding to chromatin at the site of a double-strand break. Previous studies suggested that 53BP1 acts after a ubiquitination event promoted by RNF168, although its recruitment to breaks was thought to depend only on histone H4K20 methylation. Daniel Durocher and colleagues now show that 53BP1 recruitment involves the recognition of both H4K20me2 and histone H2AK15 ubiquitination. The ubiquitin mark, and the surrounding context on histone H2A, are read by a region of 53BP1 that the authors designate the ubiquitination-dependent recruitment motif.

The inflammasome initiates innate defence and inflammatory responses by activating caspase-1 and pyroptotic cell death in myeloid cells 1 , 2 . It consists of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC. Consistent with their pro-inflammatory function, caspase-1, ASC and the inflammasome component NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis by enhancing the secretion of IL-1β and IL-18 in myeloid cells 3 , 4 , 5 – 6 . Here we show that the DNA-binding inflammasome receptor AIM2 7 , 8 , 9 – 10 has a T cell-intrinsic and inflammasome-independent role in the function of T regulatory (T reg ) cells. AIM2 is highly expressed by both human and mouse T reg cells, is induced by TGFβ, and its promoter is occupied by transcription factors that are associated with T reg cells such as RUNX1, ETS1, BCL11B and CREB. RNA sequencing, biochemical and metabolic analyses demonstrated that AIM2 attenuates AKT phosphorylation, mTOR and MYC signalling, and glycolysis, but promotes oxidative phosphorylation of lipids in T reg cells. Mechanistically, AIM2 interacts with the RACK1–PP2A phosphatase complex to restrain AKT phosphorylation. Lineage-tracing analysis demonstrates that AIM2 promotes the stability of T reg cells during inflammation. Although AIM2 is generally accepted as an inflammasome effector in myeloid cells, our results demonstrate a T cell-intrinsic role of AIM2 in restraining autoimmunity by reducing AKT–mTOR signalling and altering immune metabolism to enhance the stability of T reg cells. The inflammasome receptor AIM2 acts independently of the inflammasome to reduce autoimmunity and stabilize regulatory T cells.

In vivo and in vitro studies show that the stem-cell E3 ubiquitin ligases RNF43 and ZNRF3 act as tumour suppressors in colorectal cancer models, and are involved in the negative regulation of the cancer-associated Wnt signalling pathway through limiting the cell-surface expression of Wnt receptors. RNF43 protein in Wnt-linked colon cancer Wnt signalling is critical for the function of intestinal stem cells; it also drives colorectal tumorigenesis. Bon-Kyoung Koo et al . find that two targets of Wnt signalling, the E3 ligases RNF43 and ZNFR3, are also important negative-feedback regulators of Wnt signalling. They act by limiting the cell-surface expression of Wnt receptors. Deletion of both genes in the mouse intestine leads to expansion of LGR5 + intestinal stem cells and the development of adenomas. Furthermore, in human colon cancer cells, the expression of RNF43 reduces Wnt signalling. Mutated RNF43 has been found in human colorectal cancers, indicating that Wnt-pathway inhibitors that act at the level of Wnt secretion or Wnt-receptor activation may have therapeutic potential. LGR5 + stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals 1 . Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5 + stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5 + stem cells. In vitro , growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43 -mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.